Immunodiffusion plate apparatus

ABSTRACT

A device for the quantitative determination of protein by radial diffusion thereof through a coating of antibody on a sheet formed of a paper-thin liquid-permeable chemically inert layer (e.g., cellulose acetate) on a resilient liquid-impermeable backing (e.g., flexible plastic). The device includes a wall and marginal framework therearound, having a slot for slidably receiving a perforate or solid plate into a fixed position above the wall. The device further includes means for slidably receiving said sheet between the wall and either of the plates in position. Blood serum may be applied via a capillary tube through the openings in the perforated plate onto a sheet in predetermined placement. In one embodiment, the device contains identical backto-back units as above described. In a second embodiment, one side of such back-to-back units contains parallel members mounted on one wall and cooperating therewith to form parallel slots adapted to receive a number of sheets.

United States Patent 1 1 Feb. 29, 1972 Nerenberg [54] IMMUNODIFFUSIONPLATE APPARATUS [72] Inventor: Samuel T. Nerenberg, 21 Haynard Court,Burlingame, Calif. 94010 [22] Filed: Jan. 5, 1970 [21] Appl. No; 642

[52] US. Cl ..23/230 R, 23/230 B, 23/253 R, 195/139 LE, 424/12 [51]1nt.Cl ..C12b l/00,G0ln 33/16 [58] Field of Search ..23/230, 253, 230 B;195/139 LE [56] References Cited UNITED STATES PATENTS 3,367,841 2/1968Buissiere et al. ..23/253 X 3,389,966 6/1968 Saravis ..23/230 3,165,4501/1965 Scheidt ..l95/139 LE Primary Examiner--Morris O. Wolk AssistantExaminer-R. M. Reese Attorney-Flehr, Hohbach, Test, Albritton & Herbert[S 7] ABSTRACT A device for the quantitative determination of protein byradial diffusion thereof through a coating of antibody on a sheet formedof a paper-thin liquid-permeable chemically inert layer (e.g., celluloseacetate) on a resilient liquid-impermeable backing (e.g., flexibleplastic). The device includes a wall and marginal framework therearound,having a slot for slidably receiving a perforate or solid plate into afixed position above the wall. The device further includes means forslidably receiving said sheet between the wall and either of the platesin position. Blood scrum may be applied via a capillary tube through theopenings in the perforated plate onto a sheet in predeterminedplacement. In one embodiment, the device contains identical back-to-backunits as above described. In a second embodiment, one side of suchback-to-back units contains parallel members mounted on one wall andcooperating therewith to form parallel slots adapted to receive a numberof sheets.

8 Claims, 9 Drawing Figures PATENTED FEB 2 9 I972 SHEET 1 [IF 2INVENTOR. III." BY Samuel T." Nerenberg 74%, 9L4 w w orneys w [I m a xfi l D f IMMUNODIFFUSION PLATE APPARATUS In a method for using the abovedevice for the quantitation of protein, the antibody layered sheets areplaced into their holders in the device, the protective plate is slidinto its groove of the device to seal the coating from the air duringincubation period, the protective plate is removed and replaced with theperforate plate, blood serum is applied via a capillary tube through theperforations onto the coating, the serum and antibody reacts to formprecipitin rings and the area of said rings (representing the quantityof antigen in the blood serum) is quantitated.

In another embodiment of the device, the above antibody layered sheet isplaced at the bottom of an open-topped container, a lid is placed on thecontainer for sealing during incubation, the lid is replaced with aperforate template, serum is applied through the perforations, and theresulting precipitin rings are quantitated.

BACKGROUND OF THE INVENTION Immunological procedures for thequantitation of specific protein antigens have been widely used for bothclinical and research analyses. Such procedures are based on themeasurement of the degree of precipitation in the reaction between suchprotein antigens and their specifically reactive antibody. Perhaps themost widespread usage of this reaction is in quantitative single radialimmunodiffusion. The first such technique, performed in agar gels, wasdescribed in Mancini et al., A Single Radial Diffusion Method for theImmunological Quantitation of Proteins," 11 Prot. Biol. Fluids 370(1964). According to that technique, antibody was incorporated inuniform concentration in an agar gel with a thickness of about 1 mm.formed by means of plastic frames. After solidification of the gel intoa plate, a series of spaced-apart wells were cut therein and filled withprecise volumes of antigen solution determined by means of a calibratedmicropipette. The immunodiffusion reaction between antigen and antibodywas allowed to proceed in the gel in the fomi of a precipitin ringduring incubation for about 14 days in an oil bath. The bath functionedto prevent drying. A photographic enlarger was used to project thethus-formed circular shaped precipitin line images onto photographicpaper. These images were then cut out, weighed, and plotted on semilogpaper for quantitation. The accuracy of quantitation was found to be onthe order of i percent. The major drawback of the Mancini technique isthat the 14-day diffusion period is impractical for clinical use sincelaboratory tests normally are required within a 12-24 hour period togauge the need for immediate therapy.

In view of the pressing need for a more rapid assay procedure for theclinical determination of serum immunoglobulins, Hyland Laboratories, ofLos Angeles, California, developed a product called an Immunoplate forquantitating the three common serum immunoglobulins: 'y A Globulin(IgA), y M Globulin (IgM), and 'y o Globulin (IgG). These plates embodythe principles of Mancini but require only overnight incubation to yieldcomplete ring precipitin reactions. The ring diameters are plotted on alinear portion of semilogarithmic paper with the values of the referencesera on the coordinate logarithmic axis to obtain a standard curve.Values for the unknown samples were taken from the standard curve. Incontrast to the Mancini technique which employs precisely measuredvolumes of antigen solution in the sample wells, the Hyland technique isless accurate recommending measurement by the mere filling of wells tothe gel surface. This technique is presently being used extensively.

In 1965, an analytical study of the Hyland Immunoplates was published byFahey, .l. L., et al., in Quantitative Determination of Serumlmmunoglobulins in Antibody Agar Plates," 94 J. Immunol. 84 (1965). Theprobable error determined by Fahey et al. was about 1- percent comparedto the 1-5 percent reported by Mancini in his more refined and prolongedprocedure. ln particular Fahey, et al. calculated the standarddeviations for the three immunoglobulins to be: IgA 1 70 mg./l00 ml.,IgG 220 mg./l00 mL, and IgM 1 35 mg./ ml. Furthermore, experience in theclinical laboratory with Hyland Immunoplates, monitored by aquality-control procedure, indicate that the probable error is muchlarger than i 10 percent under actual hospital conditions, approximating20-25 percent when a number of technologists are responsible forcarrying out the procedure on a routine basis. Although fairly goodstraight-line relationships are obtained between precipitin ringdiameters and logarithmic concentration of antigen, the slopes of thecurves are very steep so that even small variations in readingprecipitin ring diameters lead to significant differences in finalresults. A further source of error is attributable to divergence of thering precipitin diameters from true circles. Although results could beimproved somewhat by multiple readings of a single precipitin ring takenin different directions, this would prolong the procedure significantlywhen numerous samples are taken. In summary,

some of the major drawbacks to the use of the Hyland Immunoplatesinclude its inaccuracy; refrigerated storage temperatures; relativelylarge volumes of undiluted serum sample (on the order of 0.25 ml.) isnecessary to determine the level of all five immunoglobulins; andinaccuracies in the system.

In order to overcome certain of the aforementioned disadvantages, C.Vergani et al. developed a technique which was published in a paperentitled Quantitative Determination of Serum Immunoglobulins by SingleRadial lmmunodiffusion on Cellulose Acetate, 4 lmmunochemistry 233(1967). According to this technique, strips of unbacked celluloseacetate membranes are utilized as the carrier medium for specificantiserum instead of the agar gel in Immunoplates. In the Verganimethod, the cellulose acetate strips are stretched between clamps toform a bridge in a moist boxlike chamber wherein diluted antiserum issprayed onto the strips. Since the antiserum soaks though the strips,they must be suspended to avoid contamination of surfaces therebelow.Following spraying, the strips are left at room temperature in the moistchamber sealed via a lid. The test sample is then applied to the stripby means of a micropipette. In order to avoid drying, the strips arethen placed into a mineral oil bath where they remain throughout theperiod of diffusion. After diffusion and the accompanying precipitinring formation the strips are removed from the oil, immersed in adetergent solution to wash off traces of oil, and washed under tapwater. The standard deviation for the results of this procedure wasstated by Vergani et al. to be comparable to that of the aforementionedFahey et al. study (:10 percent). The obvious advantages of thisprocedure as compared to agar immunodiffusion include: uniform thicknessof the cellulose acetate supporting medium which yields a sharp outlineof precipitin rings; significantly greater serum economy (e.g., a 4X28cm. strip, accommodating 50 determinations, consumes only 1.2 ml. ofdiluted antiserum); and a 2-day procedure compared to 14 days in theoriginal Mancini technique.

In spite of the aforementioned advantages, the Vergani technique has notachieved widespread use for a number of reasons. Perhaps the mostimportant is that unbacked cellulose acetate membranes are quite brittleand delicate which renders them extremely difficult to handle withoutbreaking. Furthermore, the strips lack rigidity and so must be stretchedto apply the sample accurately In addition, the strips must be suspendedon a bridge in a rather cumbersome apparatus to avoid contamination ofsurfaces therebelow since fluid will travel through the strips. Also,there is a certain loss of precision resulting from incompleteequilibration of the suspended strips and from the requirement fordirect measurement by calipers rather than use of photographicenlargement prior to performing a reading. Finally, Vergani et al.instructs that to use the strips, the antigen (e.g., blood serumcontaining immunoglobulins) must be diluted prior to application, atime-consuming step which multiplies in a procedure designed to screenthe blood serum of a large number of patients entering a hospital.

SUMMARY OF THE INVENTION AND OBJECTS This invention relates to certainholding devices for use in a single radial immunodiffusion technique ona paper-thin, liquid permeable, inert layer (e.g., cellulose acetate) ona resilient liquid-impermeable backing (e.g., flexible plastic).

In one embodiment of the invention, a holder is formed of a wall with amarginal framework on each side thereof extending outward therefrom. Theframework is provided with plate slots on the lateral sides of said walladopted to receive either a perforate template or a solid protectiveplate. Sheet slots are also provided internally of the plate slots forreceiving an immunodiffusionsheet formed of a cellulose acetate layer ona flexible plastic backing. Multiple elongated slots in parallel,'

spaced-apart relationship may be mounted on either surface of the wallfor slidably receiving a plurality of the aforementioned immunodiffusionsheets. Thus, a variety of sheet sizes may be utilized in the holder inaccordance with the number of samples selected to be run on a singlesheet (described hereinafter) resulting in a conservation of antiserum.

According to one procedural embodiment of the invention, antiserum isspread evenly onto the above immunodiffusion sheet which is theninserted, acetate layer up, via the sheet slots into the aforementionedholder. A protective plate is then slid into the plate slots above thesheet to form a compartment which protects the sheet from drying. Theantiserum is then allowed to diffuse evenly in an equilibration stage,after which the protective plate is removed from the holder and replacedwith the aforementioned template During a serum application stage,measured amounts of antigen sample (e.g., blood serum) are applied bymeans of micropipettes through the openings in the template. In adiffusion or incubation stage, the sheet is removed from Y the holderand placed under mineral oil for about 18 hours. The precipitinring-containing sheet is then washed and stained and the ring 1 imagesenlarged onto photographic paper for viewing. During thisprocedure, thebacked film is extremely easy to handle, much like photographic film.

In another embodiment of the technique and apparatus of the invention,quantitation can be performed on the above immunodiffusion sheet in abase dishlike device such as a conventional flat-bottomed Petri dish.Thus, the sheet is cut to fit snugly into the dish. The cut sheet iscoated with antiserum and placed at the bottom of the base dish, aprotective cover lid is placed thereover to protect the sheet fromdrying, and the antiserum is allowed to equilibrate. Then the lid isremoved, replaced with a template lid having a series of radially placedholes, and serum is applied. Thereafter, mineral oil is poured in thedish for coverage during incubation during which the immunodifl'usionreaction is completed without drying out of the antiserum. The remainderof the steps performed on the sheet are as described above.

It is an object of the invention to provide an improved device andtechnique for quantitating proteins by radial diffusion throughspecifically reactive antibodies which overcomes the disadvantages ofthe prior art.

It is a further object of the present invention to provide a device ofthe above type which eliminates the need for the cumbersome equipment ofthe prior art and which conserves serum and antiserum.

It is a further objection of the invention to provide a device of theabove type which decreases the time of quantitation of protein in bloodserum whereby a large number of patients may be screened simultaneously.

Additional objects and features of the invention will appear from thefollowing description in which the preferred embodiments are set forthin detail in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a top view of one embodiment ofa holder according to the invention.

FIG. 2 is a cross-sectional view of the device in FIG. 2 taken along theline 22.

FIG. 3 is a cross-sectional view of the device in FIG. 2 taken alongline 33.

FIG. 4 is an enlarged portion of the device in FIG. 3 taken in the area4-4. FIG. 5 is a top sectional view of FIG. 4 taken along the line 55.

FIG. 6 is a top view of another embodiment of a holder according to theinvention.

FIG. 7 is a cross-sectional view of the device in FIG. 6 taken along theline 7--7.

FIG. 8 is a top view of a dishlike container embodiment ac- I cording tothe invention.

FIG. 9 is a cross-sectional view of the device in FIG. 8 taken along theline 99.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring to thedrawings, rectangular immunodifi'usion sheet 11 includes a paper-thindiffusion layer 12 integral with a resilient backing 13. Layer 12 isformed of any coatable material'that is chemically inert and readilypermeable to liquid, such as cellulose acetate. Backing 13 is formed ofany suitable material that is both flexible and impermeable to liquidsuch as plastic. I have found that a particular effective material foruse as sheet 11 is a cellulose acetate layer on a backing ofpolyethylene terephthalate plastic (trademark Mylar). This integralsheet is sold under the name Titan IH-X-IOO by Helena Laboratories,Allen Park, Michigan.

Referring to FIGS. l-4, holder 14 includes facing upper and whichcooperates with wall 16 to form an upper container 20 and a lowerportion 21 cooperating with wall 17 to form a lower container 22. Sincecontainers 20 and 21 have identical construction and function, thedescription herein of container 20 applies equally to container 21 withlike part numbers assigned to each container. Holding means for sheet 11are provided at opposing edges of wall 17 and comprising in thisinstance bent portions at lateral edges 23 and 24 forming lateralgrooves 26 and 27 respectively. The forward edge of wall 17 is also bentover forming a similar groove 29. Grooves 26 and 27 serve to slidablyreceive sheet 11 at the rear edge 30 of wall 16 while groove 29functions to stop the sliding sheet in a reproducible fixed positionrelative to holder 14. A recess 31 is provided at edge 30 to facilitateremoval of sheet 11 from holder 14.

Framework 18 includes an integral frame member 32 with inwardlyextending ridges 33 on all four sides which separates wall 16 from wall17. Member 32 includes lateral sections 34 and 36, front section 37 andrear section 38. The inner surfaces of sections 34 and 36 contain slots39 and 40 respectively and lips 35 which overlap and, thus, retain wall16 in a fixed position. A flat coverplate 41 is attached to section 38,suitably by bolting to upwardly projecting ridges on the lateral sidesof section 28 to form an opening 45 therebetween communicating withslots 39 and 40. Hinged member 42 and the outer edge of section aresuitably interconnected by means of a flexible fabriclike tape 43 withadhesive backing forming the pivotal line of the hinge. A slot 44 isprovided in member 42 as a stop for a plate sliding through slots 39 and40 and opening 45. Such a plate in a stopped position would form anessentially sealed container.

Holder 14 may be formed of any rigid material such as wood, plastic ormetal. It will be apparent that the above holder construction may bevaried without altering the function thereof. For example, the entireholder could be formed of one integral structure as of a plasticmaterial.

Template 46 is of a suitable rectangular shape and size for slidingthrough opening 45 and along slots 39 and 40 and into slot 44. Template46 is provided with spaced-apart fluid application holes 47 throughwhich serum may be fed in a predetermined placement onto layer 12. Byway of example, 42 holes, 1 mm. in diameter, may be drilled through thetemplate. A handle 48 may be attached to template 46 to facilitategripping on sliding into and out of holder 14. Template may be formed ofany rigid or semirigid material, such as plastic. For visual monitoringof applied sample, as described hereinafter, the material is preferablytransparent. It is understood that although template 46 is illustratedin position in upper container 20, that interchangeably it could beplaced into lower container 22 for applying serum to a sheet 11 placedtherein.

Protective plate 49 is of approximately the same size as template 46 andmay be provided with a similar handle 50 The plate material may be rigidor semirigid. Plate 49 slides into the same slots as template 46 tocooperate with either container 20 or 22 to form chambers shielded fromsubstantial contact with surrounding air. Plate 48 is illustrated inFIGS. 1-4 to be in a reserve nonfunctional position. Plate 49 would befunctional if a second sheet 1 1 were placed in container 22.Alternatively, if template 46 is slid out and replaced with plate 49,container 20 would be sealed thereby.

Referring to FIGS. 6 and 7, an alternative holder 14a is illustrated.Only the components in holder 14a that vary from those in holder 14 willbe described in detail or assigned different part numbers. Lowercontainer 22a has the same structure and function as that of container22. On the other hand, upper container 20a functions to slidably receivethree different size sheets 11a of the same material as sheet 11 buttransverse to sheet 11. To accomplish this, three elongated members 51,52 and 53 having overhanging portions on both sides thereof are mountedin place of wall 16. The overhanging portions form slots 54 which, alongwith grooves 26 and 27, form channels for three sheets 11a. In contrastto stationary section 36, section 36a is hinged in the same manner asmember 42 to provide ready access to sheets 11a. Since these sheets 11aare received from the side, there is no reason to hinge member 42a andso it is stationary (in contrast to hinged member 42). The onlyfunctional distinction between holders 14 and 14a is the ability toutilize variable sheet sizes in the latter, thus conserving antiserumfor runs involving a small number of samples.

The foregoing description relates to a two-chambered holder with aconvenient slot for reserve storage of the template or protective platewhen not in use. It is to be understood that a single-chambered device(e.g., formed of the upper or lower half of the holder 14 or 14a) whenused for quantitating proteins on a sheet similar to sheet 11 isconsidered to be within the scope of my invention.

In one embodiment of the technique of the present invention performedeither on holder 14 or 140, large sheets of cellulose acetate backedwith Mylar plastic (commercially available as Titan III-X-lOO fromHelena Laboratories, Allen Park, Michigan) were cut into rectangularsegments to fit slots 20 of holder 11. The upper left-hand corner may benotched or cut off for orientation purposes.

In a saturation step, monospecific antibody is spread across thecellulose acetate by suitable means such as a plastic disposable razorwith the blade removable. The antibody is reactive with a specificprotein to produce a precipitin. For purposes of this description,commercial antisera monospecifically reactive with the threeimmunoglobulins, lgG, IgA, and IgM of blood serum are used as theantibody for the quantitative determination of the same immunoglobulin.The antisera are preferably diluted with saline solution prior toapplication to the cellulose acetate. The optimum dilution (i.e., theone producing the heaviest precipitin rings) must be determined for eachcommercial antiserum batch by trial and error since there are certainvariations from batch to batch. By way of example, it has been foundthat using antisera obtained from l-Iyland Laboratories, Los Angeles,California, a dilution with a 0.9 percent saline solution is effectivein the following ratios of antiserum to diluent; lgA 1:10, lgG 1:20, andIgM 1:10. The diluted antiserum is then spread uniformly onto animmunodiffusion sheet as with a plastic razor with its blade removed. Asa quantitative example, 1.5 ml. would be suffcient quantity for a 4X5inch sheet.

In an equilibration step, the coated sheet is slid into grooves 26 and27 of holder 14 by pivoting member 42 out of the way. Protective plateis then slid into its guiding slots thereover into the slot of member 42in a seated position. Thus, antiserum is prevented from drying duringequilibration or uniform saturation throughout the cellulose acetatelayer. Equilibration takes approximately 10-15 minutes at roomtemperature or about 5-10 minutes at 37 C.

The reason that equilibration may be performed in efficient holder 14 isthat backing 13 of sheet 11 is impermeable to liquid and so there is nosoaking therethrough onto wall 16. This is a marked contrast to theunbacked cellulose acetate membrane of Vergani which necessitates theuse of a cumbersome apparatus and technique for quantitation.

Following equilibration, the protective plate is removed from the holderand the surfaces of layer 12 may be inspected by an oblique light. If anirregular layer of superficial fluid is seen on the surface, that isallowed to evaporate by leaving the sheet exposed to air forapproximately 3 to 5 minutes. If the layer of fluid is unusually heavy,it may be spread more uniformly as with the plastic razor and allowed toevaporate. Drying of focal areas should be avoided to prevent distortionof the ultimate precipitin rings. Excess drying is recognizable by theappearance of occasional opaque, white spots on a dull grey background.If drying occurs, the oblique spots may be moistened with a drop ofantiserum and uniformly spread with the plastic razor.

In preparation for a serum application step, a transparent template 46is inserted into holder 14 via grooves 26 and 27. The template preventsrapid drying during application of the blood serum sample. Concentratedreference samples of each immunoglobulin are diluted for application toform three precipitins for construction of standard curves in theanticipated range of sample values as explained hereinafter.

Referring to FIG. 5, in decreasing order of concentration points 56 a-crepresent applied reference sera and the corresponding ultimatelystained precipitin rings are represented at 57a-c. In like manner,points 58a-c represent applied unknown sera and precipitin rings 59a-crepresent corresponding stained precipitin rings. Sera, both referenceand unknown samples, may be labeled with bromphenol blue dye by mixing afew grains of dye powder with each sample for easier viewing of theprecipitin.

Sample is applied through the template holes suitably by means of a tubewhich accurately measures small quantities of fluid. A preferredapplicator is a microcapillary tube holder fitted with disposable 0.5p.l self-filling capillary tube such as the Drummond type sold by HelenaLaboratories, Allen Park, Michigan. For the most accurate control ofsamples, it has been found that mouth pressure exerted through narrowgauge tubing is more effective in forcing the sample from the capillarytubes than is the use of a compressible rubber bulb as supplied with theDrummond capillary tube holder. Sample in the microcapillary tubes isapplied through template holes directly onto the cellulose acetatesurfaces. During application of the dye-colored samples, total emptyingof each capillary tube was determined by monitoring or viewing throughthe transparent plastic template. Each sample forms a concentricblue-colored spot on the surface of the cellulose acetate sheet.

For incubation, the template is slid out of the holder, member 42 ispivoted outwardly, and sheet 11 is removed from the holder and placedunder the mineral oil. The sheet is then incubated overnight (e.g.,about 18 hours) suitably at the following temperatures: about 25 C. forlgA, about 410 C. for lgG, and about 37 C. for IgM. The purpose of themineral oil is to shield the sheet from drying out during incubation. Itis noted that if holder 14 could be completely air sealed with theprotective plate, incubation could be performed directly in the holder.

Following incubation, the sheet is removed from the mineral oil andsuspended as from a clamp for a few minutes to allow residual oil todrip free of the sheet. In order to remove any remaining oil, the sheetmay then be washed in a detergent-saline solution for approximatelyminutes. In a second wash the sheet may be retained in a 0.9 percentsaline solution for approximately 30 minutes to remove unreactedproteins. The saline solution can be removed from these sheets byrinsing in tap water. To render the precipitin rings clearly visible,they may be stained as with Thiazine Red in a 1 percent acetic acidsolution for approximately 30 minutes followed by rinsing as with 1percent acetic acid solution. After allowing the sheets to dry, thestained precipitin rings represented at 57 and 59 are readily visible.

To quantitate the precipitin, it is preferred to project the driedunstained cellulose acetate sheets through a standard photographicenlarger for final viewing on photographic paper. In one system, theimages projected onto sensitized paper such as Ectamatic SC paperproduced by Eastman Kodak Co. and then passed through an EctamaticProcessor which provides enlarged images on a fixed photographic paperin a semidried state in less thana minute. The photographic paper maythen be removed from the dark room for analysis. Diameters of eachprecipitin ring may be compared in several directions with a compass toconfirm the formation of true circles. If so, the magnitude of the ringdiameter may be measured. 0n those infrequent occasions when the ringprecipitin do not form true circles, the area enclosed by the rings maybe cut out along with the reference areas for weighing. A standard curvemay be prepared by plotting the ring diameters and/or the weights of thepaper areas circumscribed by the precipitin rings 57 of the referencespecimens on the linear axis of two-cycle semilog graph paper againsttheir known protein values plotted on the logarithmic axis. Unknownsamples may then be read directly from the standard curve.

A statistical analysis of commercial standard reference sera for theimmunoglobulins lgA, IgG, and IgM, was performed by applying bloodsamples to antisera-coated, backed cellulose acetate sheets according tothe aforementioned technique and using reference sera as above-describedfor points of the standard curve. The results of this analysis aresummarized in Table I.

Referring to Table I, it is apparent from the above standard deviationsthat a significant improvement in precision of quantitation is achievedby the present device and technique in comparison to that of theaforementioned agar gel technique as studied by Fahey et al. withstandard deviations of IgA i 70 mg./l00 ml., lgG-220 mg./l00 ml., andIgM35 mg./l00 ml. The present invention has the same superiority ofprecision over the Vergani unbacked cellulose acetate technique sincethe latter is comparable in accuracy to that of Fahey.

An important factor in the improved accuracy of the technique of thepresent invention is the ability of the plasticbacked cellulose acetatesheet to be handled as a regular photographicfilm. Thus, projection andenlargement of the stained precipitin rings onto the photographic paperenables the enlarged photographically developed precipitin images to bemore accurately measured than precipitin rings viewed through amicroscope. It is noted that even a slight error in direct measurementof a ring diameter would introduce a relatively large error in the finalresult due to its logarithmic value.

Furthermore, recording of precipitin rings on photographic paper may beperformed almost instantaneously with great precision compared to thetedious and relatively inaccurate technique of tracing the images in adarkroom recommended by Mancini et al. In addition, the time utilizedfor processing the photographic paper (less than a minute) isexceedingly more rapid than the tracing of individual images of manysamples. Finally, increased accuracy and speed of an analysis areprovided by the use of the compass to detect distorted".

precipitin rings. It is only those infrequently occuring distorted ringswhich require the time consuming process of weighing the closed areasfor quantitation.

Referring to FIGS. 8 and 9, another embodiment is illustrated of adevice which may utilize the aforementioned backed cellulose acetatesheet in a technique analogous to that described above. This dishlikedevice includes a container dish 60, an overlaying replaceable perforatetemplate lid 61, and a solid protective lid (not shown) interchangeablewith template 61. Dish 60 is suitably a flat-bottomed, open-topped rigidor semirigid dish such as disposable plastic, commercially availablePetri dish. The size of dish 60 may vary in accordance with the numberof samples to be applied in a single run. To conserve antiserum, an mm.diameter dish 60 may be employed along with template lid 61, with 16holes to accommodate a like number of samples as described hereinafter.Template 61 (and the protective lid) may be of a flat-bottomed dishlikeshape similar to dish 60 but with a slightly larger diameter so that anessentially snug fit is created with lid 61 overlying dish 60.

A circular disc 63 of the plastic backed, cellulose acetate materialdescribed above may be formed to conform to the inner diameter of dish60. A spot may be applied at one point on the edge of the dish to orientthe application of samples. Discs 63 may be stored in individual dishes60 ready for use.

Application of antiserum may be performed as described in theaforementioned technique. The equilibration may be performed by placingdisc 63 into dish 60 and covering the dish with protective lid. Theprecautions to prevent drying are described above.

Referring to FIG. 8, template lid 61 may be drilled with a series ofholes 64 in a spaced-apart relationship. This spacing is planned so thatwhen sample is applied through holes 64 onto disc 63, there issufficient clearance for the spreading of each precipitin ring withoutcontact with adjacent rings. Although the dishlike device is describedwith reference to a circular walled dish, it is to be understood thatother shapes, such as rectangular, may be employed to the same effect.

In the incubation stage, template lid 61 may be removed and mineral oildirectly spread over disc 63 in dish 60. The timing of incubation is asdescribed above. The remainder of the quantitation procedure includingwashing, staining, and projecting the results onto photographic paper,may also be performed as described above.

Quantitating by means of holders 14 or 14a has certain advantages overquantitation with dish 60. For example, specimen spacing, to preventoverlapping of precipitin rings, is easier to plan on a rectangulartemplate and the corrolation of precipitin rings with the properspecimens is also simpler with a rectangular shape. Furthermore, thedisc shaped sheet is somewhat more difficult to handle during aprocedure, e.g., during removal from dish 60.

It will beapparent from the foregoing that I have provided a number ofembodiments of a holding device with which accurate and simpleimmunodiffusion quantitations may be performed on a flexible plasticsheet covered with a cellulose acetate film. Quantitation according tothis invention is exceedingly accurate, easy and rapid to perform, andeconomical of serum and antiserum. Although the invention is describedwith respect to specifically constructed holders, it is understood thatequivalent holders are within the present scope.

We claim:

1. In a method for the quantitative determination of protein, the stepsof layering antibody specific to said protein onto a paper-thincellulose acetate coating on a liquid impermeable resilient backing,placing said backed layered coating into a frame, sliding a protectiveplate into a groove in said frame, sealing said coating from the air,incubating said sealed coating for a sufficient time to cause theantiserum to be uniformly absorbed into the coating, removing saidprotective plate from said groove, sliding into said groove a perforateplate having a plurality of openings in spaced-apart relation, applyingan unknown protein antigen through said template to said coating,reacting said antibody and antigen on said coating to form precipitinrings, and quantitating the area of said rings.

2. A device for the quantitative determination of protein by radialdiffusion comprising open-topped containermeans, a sheet disposed insaid container means, said sheet being formed of a cellulose acetatelayer on a resilient impermeable backing, said layer being saturatedwith antibody specifically reactive with said protein, a perforatetemplate adapted to fit over said container means, said template havingholes in spaced-apart relation for applying protein sample to said layerin a predetermined pattern.

3. In a device for the quantitative determination of protein by radialdiffusion thereof through a coating of antibody specifically reactivewith the protein, a sheet formed of a paper-thin liquid-permeablechemically inert layer on a resilient liquid-impermeable backing, wallmeans, means forming a marginal framework on each edge of said wallmeans extending outwardly therefrom, said framework means having slotmeans adapted to slidably receive a plate, holding means disposed atlateral edges of said wall means at a level between said wall means andsaid slot means, said sheet being inserted in said holding means.

4. A device as in claim 3 wherein said coating is formed of a celluloseacetate and said backing is comprised of a pliable plastic.

5. A device as in claim 3 together with a perforate plate having aplurality of openings in spaced apart relationship and slidably receivedin said slot means whereby liquid sample may be applied via a tubethrough said perforations onto said sheet in predetermined placement.

6. A device as in claim 5 wherein said sheet is disposed between saidwall means and said perforate plate.

7. A device as in claim 3 including a solid protective plate slideablyreceived in said slot means, said marginal framework means, wall meansand protective plate cooperating to form an air-sealed compartment forsaid sheet.

8. A device as in claim 7 wherein said sheet is disposed between saidwall means and said protective plate within said air-sealed compartment.

2. A device for the quantitative determination of protein by radialdiffusion comprising open-topped container means, a sheet disposed insaid container means, said sheet being formed of a cellulose acetatelayer on a resilient impermeable backing, said layer being saturatedwith antibody specifically reactive with said protein, a perforatetemplate adapted to fit over said container means, said template havingholes in spaced-apart relation for applying protein sample to said layerin a predetermined pattern.
 3. In a device for the quantitativedetermination of protein by radial diffusion thereof through a coatingof antibody specifically reactive with the protein, a sheet formed of apaper-thin liquid-permeable chemically inert layer on a resilientliquid-impermeable backing, wall means, means forming a marginalframework on each edge of said wall means extending outwardly therefrom,said framework means having slot means adapted to slidably receive aplate, holding means disposed at lateral edges of said wall means at alevel between said wall means and said slot means, said sheet beinginserted in said holding means.
 4. A device as in claim 3 wherein saidcoating is formed of a cellulose acetate and said backing is comprisedof a pliable plastic.
 5. A device as in claim 3 together with aperforate plate having a plurality of openings in spaced apartrelationship and slidably received in said slot means whereby liquidsample may be applied via a tube through said perforations onto saidsheet in predetermined placement.
 6. A device as in claim 5 wherein saidsheet is disposed between said wall means and said perforate plate.
 7. Adevice as in claim 3 including a solid protective plate slideablyreceived in said slot means, said marginal framework means, wall meansand protective plate cooperating to form an air-sealed compartment forsaid sheet.
 8. A device as in claim 7 wherein said sheet is disposedbetween said wall means and said protective plate within said air-sealedcompartment.